Toll-like receptors (TLRs) are pattern recognition receptors that bind to pathogenic molecules or danger molecules to trigger an innate immune response. Several of these receptors reside intracellularly, including TLR9, which is primarily located at the endoplasmic reticulum in unstressed or uninfected cells. De Leo et al. identified a role for TLR9 in sustaining autophagic flux in stressed cells. Autophagy and lysosomal function are critical for cell health. Indeed, mutations in the gene encoding the phosphatidylinositol (4,5)P2 phosphatase OCRL cause Lowe syndrome and lysosomal dysfunction. Depleting kidney proximal tubule cells (PTCs) of TLR9, but not of other intracellularly localized TLRs, reduced the colocalization of the following proteins and lipids with the lysosomal marker LAMP1 in response to the autophagic stimulus of nutrient and growth factor starvation: OCRL, the adaptor protein AP2, the phosphatidylinositol 5P kinases PIP5K1α and PIP5K1β, and PI(4,5)P2. Knocking down or pharmacologically inhibiting TLR9 resulted in the accumulation of autophagosomes and enlarged lysosomes, impaired lysosome-autophagosome fusion, and reduced the activation and nuclear translocation of the inflammatory transcription factor NF-κB. TLR9 translocated to the lysosomal compartment in starved cells through a process dependent on the chaperone UNC93B1. Because these cells were not infected, an endogenous cellular ligand for TLR9 mediated this translocation. Induction of mitochondrial damage (without starvation) stimulated this translocation of TLR9 and the recruitment of OCRL, AP2, and PIP(4,5)P2; whereas depleting mitochondrial DNA reduced this response in starved cells. Similar to knock down or impairment of TLR9 signaling, knocking down OCRL or pharmacological inhibition of OCRL also reduced autophagic flux and autophagosome-lysosome fusion, which resulted in the accumulation of autophagosomes even in nutrient-replete conditions. Catalytically inactive OCRL did not rescue autophagic flux in the OCRL knockdown cells, but knocking down PIP5K1α and PIP5K1β did, indicating that the abundance of PI(4,5)P2 played a key role in regulating autophagosome-lysosome fusion. PI(4,5)P2 inhibits the lysosomally localized calcium channel MCOLN1 (also known as TRPML1), and MCOLN1 exhibited a reduced calcium release response when the OCRL knockdown cells were exposed to an MCOLN1 agonist. Lowe syndrome patients also exhibited an accumulation of autophagosomes in proximal kidney tubule biopsies, and pharmacological activation of MCOLN1 increased autophagic flux in PTCs from Lowe syndrome patients. The authors propose that mitochondrial DNA released through lysosomal or autophagosomal activity binds to TLR9, stimulating its translocation to the lysosome, where it recruits various proteins including OCRL, which reduces the abundance of PI(4,5)P2 thus enabling lysosome-autophagosome fusion and autophagic flux. This process is important both for homeostatic autophagy and for autophagy induced by cell stress, such as nutrient and growth factor deprivation.